Cigarette smoking causes 30% of all cancer mortality in the developed world. Tobacco-specific nitrosamines, the subject of this proposal, are accepted causes of cancer in people who use tobacco products. The two most carcinogenic of these compounds- 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN)- are known human carcinogens according to IARC. Our laboratory is the acknowledged world leader in studies on mechanisms of tobacco-specific nitrosamine carcinogenicity, research that has been continually funded by NCI grants since 1976, including the current MERIT award. Our overall goal is to understand mechanisms by which tobacco products cause cancer and use this information to develop new approaches to tobacco control and prevention of tobacco-induced cancer. The current project period of this grant, which began in 2004, has been exceptionally productive. Twenty-one original peer-reviewed manuscripts and 12 reviews/book chapters have been published or submitted as of June, 2007. A full one page abstract and progress report are found on pages 19-29 of this application. We propose to continue this research with the following broad objectives: 1. Extend our new MS methods for DMA adduct analysis to include the pyridylhydroxybutyl and methyl adducts of NNAL and the 5'- hydroxylation adducts of NNN and improve the sensitivity of these methods. 2. Improve sensitivity and high throughput for measurement of the NNK metabolite NNAL in human blood and urine, and apply these methods in nested case control studies in which pre-diagnosis samples have been stored from smokers who developed lung cancer and from matched controls. 3. Identify the receptor(s) in the rat lung that specifically binds the NNK metabolite (S)-NNAL 4. Further develop the toenail biomarkers for NNAL and cotinine. 5. Determine the structure of an inhibitor of carcinogen metabolic activation, which we have identified in human lung microsomes. 6. Continue our research on inter-individual differences in human metabolic activation and detoxification of tobacco-specific nitrosamines, and on the endogenous formation of these carcinogens in humans. The results of these studies should provide important new data which will be critical for developing new approaches to the prevention of tobacco-induced cancer.